Attempts were made to define which fatty acid (2∶0 to 18∶1) was optimally oxidized by isolated colonocytes (colonic epithelial cells) and to select inhibitors of fatty acid oxidation which would be analogous in their action to the inhibition of fatty acid oxidation observed in colonocytes involved with ulcerative colitis. Isolated colonic epithelial cells of Sprague‐Dawley rats were used with 2‐mercaptoacetate, dichloroacetate, 3‐mercaptopropionate, 4‐mercaptobutyrate, 4‐sulfatebutyrate, 2‐bromobutyrate, sulfite ions and nitrite ions. n‐Butyrate (4∶0) was maximmaly oxidized to CO₂ and ketone bodies (mean value 5.46 μmol/min/g dry wt). Oxidation of butyrate to CO₂ was diminished by 2‐bromobutyrate, sulfite ions and all mercapto fatty acids. Both fatty acid oxidation and glucose oxidation were significantly inhibited by 2‐bromobutyrate, while mercapto fatty acids and sulfite inhibited fatty acid oxidation (p<0.01) without significantly changing glucose oxidation. Observation with 2‐mercaptoacetate and sulfite correlate with early changes of fatty acid oxidation observed in cases of ulcerative colitis, and warrant further study with isolated colonocytes of man.